The present invention provides a method of sulfurizing phosphorus containing compounds, which is reliably useful and which can be used when automating the preparation of such compounds.
The method of the present invention is applicable to a wide variety of compounds, even though it initially grew out of a desire to provide a suitable methodology for the sulfurization of oligonucleotide, which lacked drawbacks associated with other methods generally utilized by those skilled in the art.
The current methodology for the sulfurization of oligonucleotide phosphite triesters via standard "phosphoramidite" chemistry uses a solution of elemental sulfur (S.sub.8) in carbon disulfide: pyridine, triethylamine (1:1:0.1 ) (Stec, W. J. et al, J. Am. Chem. Soc. 106, 6077-79, (1984)). The sulfurization reaction with the reagent is slow (ca. 7.5 min) (Stein, C. A. et al, Nucl. Acids Res. 16, 3209-21, (1988)) and because of the insolubility of elemental sulfur in most organic solvents, its precipitation in the delivery lines of commercial instruments, invariably occurs and causes instrument failure. Consequently, the automation of sulfurization reaction with elemental sulfur is erratic and unreliable.
In an attempt to alleviate problems with unreliability and erraticacy, a "nucleoside-H-phosphomate" chemical approach has recently been suggested in the automated preparation of oligonucleotides (Garegg, P. J. et al, Chem. Scripta 28, 280 (1985); Froehler, B. C. et al, Nucl. Acids Res. 14, 5399 (1986)). The method employs sulfurization with elemental sulfur in one step outside the instrument (Froehler, B. C., Tetrahedron Lett. 27, 5565-68 (1986)). Although the automation of the sulfurization reaction is not required with this approach, the "nucleoside-H-phosphomate" methodology suffers from lower stepwise yields (94-96%) during the preparation of synthetic oligomers relative to the "phosphoramidite" approach, which, consistently generates stepwise yields of ca. 99%. This consideration is important especially when large amounts of pure synthetic oligomers are required. Most importantly, the sulfurization of predetermined phosphorus moieties within an oligomer cannot yet be achieved by the "nucleo-H-phosphomate" method.